by Pavel IVANOV, Cand. Sc. (Medicine), leading researcher, N. Blokhin Russian Oncological Research Center, RAS;
Radiy MAKHLIN, Cand. Sc. (Technology), laboratory head, Research Institute of Precision Engineering;
Nikolai MOSHECHKOV, head of a design team at the same Institute;
and lAlexander KHINIKADZE director of the same Institute
Cancer, the Number Two killer after cardiovascular diseases, has been the target of vigorous struggle throughout the twentieth century. Within the last few decades medics have developed a battery of antitumor preparations for quite hopeless cases. Most of these medications belong to the group of cytotoxic agents, i.e. those destroying the actively dividing cells. Such drugs, when used in effective doses, suppress the further spread of malignancies and prevent secondary cancers, the metastases.
And yet... Such potent cytotoxic drugs injure sound tissues-first and foremost, the actively dividing bone marrow cells responsible for hematogenesis, or blood formation. The death of these cells has an adverse effect on the production of fresh blood cells and thus lowers the immunity of the organism; it upsets homeostasis (natural balance) and is conducive to anemia. The effect is similar to that of high- dosage radiation at nuclear facilities (acute radiation disease) or in the course of radiotherapy.
Therefore scientists in many countries have been searching after ways of protecting bone marrow cells during chemotherapy or else of their subsequent rehabilitation. The idea of a donor's marrow transplant (allogenic transplantation) was not at all new. In fact, all the way back in 1891 French doctors made the first attempt to use a bone marrow substrate for treatment but failed in their endeavor. True, the preparation was administered orally, that is through the mouth. And the attempt of German medics in 1937 to try intramuscular injections was not successful either. A decade later, in the mid-1940s, one tried allogenic transplantation performed intravenously. Success came only in 1968 when American doctors could cure the first cancer patient by this very method.
Now we know: this method has drawbacks. The point is that the organism does not take bone marrow from any donor. As a rule, the transplant is rejected, and the outcome is lethal. So it is essential to have a vast databank on the "curative substance", so to speak; but even this costly procedure does not give us a sure-fire guarantee.
Yet another way of coping with the problem of hematoxicity (blood poisoning) is the now widespread method of autotransplantation when a patient donates his own bone marrow. A definite amount of marrow (250 to 400 g) is taken and preserved for a time under deep-freeze conditions until a course of chemotherapy has been completed. Thereupon the bone marrow substrate is injected intravenously. The transplanted hematogenetic (blood-producing) cells will keep proliferating, and the blood system function will be restored to normal after a time. This method proved effective, especially for child patients, with as much as 70 percent of the cases saved given an early diagnosis.
But even this method is not devoid of shortcomings either. Since chemotherapy destroys both malignant and blood-producing cells (red blood cells and blood granulocytes), the natural immunity of the organism to various viruses falls precipitously. Strict precautions become necessary to protect the patient against viral infection: he is taken to a sterile intensive-care unit (ICU) and given high-cost antibiotics. Stringent aseptic rules are a must. This course of treatment takes two or three weeks, for the number of truncal cells within the bone marrow is small-just about 3 percent of the total mass. That is why the rehabilitation of the blood system and of immunity proceeds slowly.
Furthermore, since in most cases the patient's bone marrow is riddled with malignant cells (micrometastases), its transplantation makes no sense in the long run-a relapse of cancer is inevitable. So, the circle comes full swing, it would seem. But there is still a glimmer of hope: Why not cleanse the bone marrow transplant from cancer cells?
Such experiments have been carried out in many countries, with a variety of methods tried: in particular, filtration, differential centrifugation, chromatography and other techniques. One method has been found to be best: separation of cells by means of specific immunosorption-magnetoguided sorbents that make it possible to segregate cells in a magnetic field. Now, a closer look.
Magnetoguided sorbents are composed of ferromagnetic particles (metal pellets not more than 5 urn in diameter coated with polystyrene) bonded with monoclonal antibodies (monoclonals). The latter are capable of attracting either sound or malignant cells. Accordingly, we obtain positive and negative immunosorption. So the cells belonging to either group will be entrapped (for each attracts three or four antibodies). And should we place the thus treated bone marrow cells into a magnetic field, they will be segregated into "good" and "bad" ones. Upon elimination of magnetoguided sorbents
the "goodies" and the "baddies" are collected into separate receptacles. The healthy cells are stored this way until transplantation.
This procedure enables us to get rid of micrometastases and prevent the recurrence of malignancies. And the number of truncal cells within the autotransplant will increase considerably as a result. All that will whip up the blood regeneration process and, as a consequence, shorten the time of ICU treatment.
Corresponding bone-marrow cleansing separators have been designed in the United States, with Britain, France and Norway contributing certain essential materials. Yet their technique involves exorbitant costs: a patient has to shell out 100,000 to 250,000 dollars for the operation. What with the cleansing efficiency not above 70 percent, the cure guarantee is not high enough. Besides, the processed truncal cells are often deformed and eliminated as no good. There are other shortfalls as well.
That's where experts of the Central Research Institute of Precision Engineering (the town of Klimovsk) decided to step in: in 1993 they got down to the job of designing a better magnetic separator of bone marrow cells. They could rely on the hands-on experience of six years in designing medical technology. The RF Ministry of Public Health upheld the initiative. Other big research centers joined hands on the project: the N. Blokhin Russian Oncological Research Center, the MedBioSpektr R&D Center and the State Research Institute of Chemistry and Technology of Heteroorganic Compounds.
Research teams had to attack several basic problems-namely, design a proper bone- marrow cleansing technology and the hardware, i.e. the separator itself. Furthermore, the designers were not satisfied with the then available immunosorbents. And ultimately they built the desired magnetic separator of bone marrow cells.aMSK-1.
The main part of this apparatus is a container with a physiologic salt solution-that's where the bone marrow transplant and the immunomagnetic sorbent are placed. In case of positive selection, the sorbent binds to sound cells in the incubation mixture; and if the selection is negative, it binds to malignant cells. Next comes the step of magnetic separation, a process of cells segregation. Then the contaminated part of the incubation mixture is decanted, and the solution is supplemented with a clean mixture back to the initial volume; the same procedure is then repeated twice. In this wise we get a fantastic degree of purification, close to 90 percent.
The next step is to liberate the cells captured by the immunomagnetic sorbent. For this purpose an enzyme cleaving their bonds is added to the incubation mixture, and the container is moved into a magnetic field again. Thereupon the healthy cells are poured off into a special vessel for immediate use or conservation.
The custom-fitted MSK-1 apparatus measures 600 mm in length, 400 mm in width and 500 mm in height. Its weight is 25 kg. It is powered by the ordinary AC network of 220 V, and the power consumed is equal to 250 W. In these characteristics MSK-1 may not stand out from the group of its foreign counterparts. Yet there is something else to make it without peer.
The MSK-1 purification efficiency is about 90 percent against 70 percent with its foreign analogs. It does not deform truncal cells. The whole process takes just 100 min to cleanse 250 g of the marrow substrate. The processor-controlled reaction proceeds in the automatic mode. The magnetoguided capsules are bound to truncal cells at a constant preassigned temperature in keeping with sterility conditions. And our bone marrow cleanser is simpler in design and costs less than foreign-designed separators.
As MSK-1 was still in the gestation stage, research scientists tangled with a formidable job of designing immunosorbents. Let us remember that these are a
complex of ferromagnetic particles or capsules and monoclonals. We had to start from scratch since Russia had never produced particles of either type.
To begin with, our research teams had to sort out the available ferromaterials and select those that could be made into ~5 um pellets or capsules possessing the required level of magnetization. These corpuscles were to be coated with surfactants, and inert or specialized substances with prescribed characteristics. And last, magnetoguided capsules had to be capable of forming tight bonds with antibodies.
After many experiments we chose polystyrene-coated iron. This conglomerate satisfies the above requirements and besides, possesses high sorption properties: it can be sterilized and kept for a long time in buffer solutions. This point is of the utmost importance, for immunosorbents made from it may be badly needed at a later date.
And finally, monoclonal antibodies, the monoclonals. Recall that they are attracted by one particular type of cells and make subsequent cell separation possible. The production of monoclonals is a very tricky biochemical process that takes in several steps followed by purification performed by most up-to-date methods (e.g., affinity chromatography). Our researchers have negotiated all the obstacles, and today our country has developed an array of marrow-cleansing antibodies in more than 50 varieties.
MSK-1, the brainchild of Russian science and engineering, is a real breakthrough in the struggle against oncodiseases. MSK-1 offers unique possibilities for cancer therapy and costs 0.3 to 0.25 as much as foreign counterparts. The apparatus has gone through a series of human trials. What we need now is mass production and wide- scale use in clinical practice. Besides, MSK-1 and a kit of immunomagnetic sorbents will enable medics not only to enhance the efficiency of treatment of malignancies and blood diseases. It will also allow to build up cryobanks of bone marrow cells for workers facing radioactive or chemical hazards (those employed at nuclear power stations and related facilities, rescue and emergency teams, troubleshooters at nuclear objects and the like).
MSK-1 offers many other advantages in areas like virology, gene engineering and in search of HIV treatment methods.
The innovative apparatus has been patented in the Russian Federation.